Water deficit tolerance in sugarcane is dependent on the accumulation of sugar in the leaf

Sugarcane productivity is severely affected by the occurrence of water deficit in the field, causing inhibition of growth and sugar production. Evaluating physiological responses of sugarcane under water deficit conditions is essential to understand physiological variables responsible for reaching homeostasis. Therefore, we analysed physiological traits of two sugarcane genotypes, RB835486 (Tolerant) and RB855453 (Susceptible), under water deficit conditions: well-watered (WW-Control), water deficit (WD) and rewatered (RW). The physiological response was evaluated using linear regression and multivariate analysis. Some characteristics such as water potential in leaves, photosynthesis, chlorophyll fluorescence, chlorophyll index, sucrose and starch contents did not show differences between the genotypes under water deficit conditions. However, the tolerant genotype showed increased reducing sugars content in the leaves, whereas the susceptible genotype had increased non-photochemical quenching (qN). After rewatering, the susceptible sugarcane genotype showed higher electron transport rate (ETR) and efficiency of PSII (Y). Multivariate analysis revealed that non-photochemical quenching and reducing sugars in the leaves were physiological variables responsible for reaching homestasis under water deficit conditions. Therefore, the reducing sugars concentration should be considered a physiological variable responsible for the adjustment made by the tolerant sugarcane genotype when submitted to water deficit.     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.     

Feeding behavior of Brevicoryne brassicae in resistant and susceptible collard greens genotypes: interactions among morphological and chemical factors

The cabbage aphid, Brevicoryne brassicae (L.) (Hemiptera: Aphididae), is distributed throughout the tropical and subtropical areas of the world. The main crops attacked by B. brassicae are cabbage, collard greens, broccoli, Brussels sprouts, and cauliflower. To survive the attack of pest insects, plants have evolved various resistance mechanisms that may affect pest feeding behavior. The use of electronic monitoring through EPG (electrical penetration graph) can help characterize and distinguish the resistance mechanisms involved. This study evaluated the feeding behavior of B. brassicae in eight genotypes of collard greens, Brassica oleraceae L. var. acephala (Brassicaceae), exhibiting antixenosis and/or antibiosis resistance to this insect. Possible correlations were established between the glucosinolate levels, the hardness, and the epicuticular wax on the leaves vs. aphid feeding behavior. On the genotypes 22V, 5E, and 27VA, for which many ‘potential drop’ waves were performed, aphid development was slower, indicating antixenosis as resistance type. Aphids on the genotypes 22V and 24X required more time until accessing the phloem, also suggesting antixenosis as resistance category. Genotypes 22V and PE had hard leaves, which also points at antixenosis. Genotypes 20T and HS had higher total wax and wax mg⁻¹. Feeding parameters on ARI and 24X were similar to those observed on HS; antibiosis is likely to be the predominant resistance category of this germplasm. Because HS was considered as a susceptible standard genotype in this study, a higher gluconapin amount indicates that this compound does not influence cabbage aphid feeding behavior. The present study confirms that analysis of the physical and chemical aspects of collard greens genotypes by the EPG technique can provide a useful approach for the study of plant resistance to cabbage aphids.     

Loss and rescue of osteocalcin and osteopontin modulate osteogenic and angiogenic features of mesenchymal stem/stromal cells

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.     

Role of Sonic hedgehog signaling in cell cycle,oxidative stress,and autophagy of temozolomide resistant glioblastoma

The first-line chemotherapy treatment for Glioblastoma (GBM) - the most aggressive and frequent brain tumor - is temozolomide (TMZ). The Sonic hedgehog (SHH) pathway is involved with GBM tumorigenesis and TMZ chemoresistance. The role of SHH pathway inhibition in the potentiation of TMZ's effects using T98G, U251, and GBM11 cell lines is investigated herein. The combination of GANT-61 and TMZ over 72 hr suggested a synergistic effect. All TMZ-resistant cell lines displayed a significant decrease in cell viability, increased DNA fragmentation and loss of membrane integrity. For T98G cells, G₂/M arrest was observed, while U251 cells presented a significant increase in reactive oxygen species production and catalase activity. All the cell lines presented acidic vesicles formation correlated to Beclin-1 overexpression. The combined treatment also enhanced GLI1 expression, indicating the presence of select resistant cells. The selective inhibition of the SHH pathway potentiated the cytotoxic effect of TMZ, thus becoming a promising in vitro strategy for GBM treatment.     

A dynamic model linking cell growth to intracellular metabolism and extracellular by-product accumulation

Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies.     

Current genetic engineering strategies for the production of antihypertensive ACEI peptides

Hypertension is a major and highly prevalent risk factor for various diseases. Among the most frequently prescribed antihypertensive first-line drugs are synthetic angiotensin I-converting enzyme inhibitors (ACEI). However, since  their use in hypertension therapy has been linked to various side effects, interest in the application of food-derived ACEI peptides (ACEIp) as antihypertensive agents is rapidly growing. Although promising, the industrial production of ACEIp through conventional methods such as chemical synthesis or enzymatic hydrolysis of food proteins has been proven troublesome. We here provide an overview of current antihypertensive therapeutics, focusing on ACEI, and illustrate how biotechnology and bioengineering can overcome the limitations of ACEIp large-scale production. Latest advances in ACEIp research and current genetic engineering-based strategies for heterologous production of ACEIp (and precursors) are also presented. Cloning approaches include tandem repeats of single ACEIp, ACEIp fusion to proteins/polypeptides, joining multivariate ACEIp into bioactive polypeptides, and producing ACEIp-containing modified plant storage proteins. Although bacteria have been privileged ACEIp heterologous hosts, particularly when testing for new genetic engineering strategies, plants and microalgae-based platforms are now emerging. Besides being generally safer, cost-effective and scalable, these “pharming” platforms can perform therelevant posttranslational modifications and produce (and eventually deliver) biologically active protein/peptide-based antihypertensive medicines.